Project Summary/Abstract There has been little change in mortality rate of human colorectal cancer (CRC) in the past decades and the treatment options available for CRC are still very limited today. In CRC and most other human cancers, the tumor suppressor TP53 gene is frequently inactivated by mutation or deletion. Consequently, tremendous effort has been made to restore the p53 activity for cancer therapy. However, no p53-based therapy has been successfully translated into clinical cancer treatment due to the complexity of p53 signaling. Therefore, instead of restoring the p53 activity, identifying vulnerabilities conferred by TP53 deletion or mutation is a novel strategy for fighting against the p53 deficiency in human cancer. In a recent study published in Nature, we confirmed that genomic deletion of TP53 is frequently accompanied by the partial loss of neighboring essential genes and cancer cells with hemizygous TP53 deletion are remarkably vulnerable to further suppression of such neighboring essential genes. We revealed that POLR2A is such an essential gene that is often partially co-deleted with TP53 in human cancers. Such hemizygous loss of TP53/POLR2A occurs in 53, 60, and 41% of colorectal, breast, and pancreatic cancers, respectively. The POLR2A activity is specifically inhibited by ?-Amanitin (Ama), a cyclic peptide of 8 amino acids found in the mushroom Amanita phalloides. We demonstrate that low doses of Ama conjugated with anti-epithelial cell adhesion molecule (EpCAM) antibody for targeting CRC can result in much enhanced tumor regression in murine models of human CRC with hemizygous deletion of POLR2A without evident systemic toxicity. However, a small fraction of CRC cells are found to be resistant to Ama in a dose-dependent manner. These drug-resistant cells are often called cancer stem-like cells (CSCs) or tumor initiating cells (TICs). A common feature of the CSCs in many cancers (e.g., CRC together with breast and pancreatic cancers) is that they overexpress the variant CD44. Our preliminary data show that the Ama-resistant CRC cells are indeed enriched with CD44, but not EpCAM. Moreover, our studies show that human breast and prostate CSCs can be effectively destroyed in vitro and in vivo using anticancer agent-laden nanoparticles that target the variant CD44 (but not the normal or non-variant CD44 on normal stem cells) with no evident systemic toxicity. Here, we hypothesize that targeted delivery of Ama and/or clinically used anticancer drugs to the variant CD44+ cancer cells with nanoparticles can overcome their drug resistance. We will test this hypothesis with two specific aims: 1, to determine the mechanisms of resistance to the Ama-based POLR2A-targted therapy of human CRCs with hemizygous loss of TP53 and 2, to determine if the combined therapy of Ama and clinically used anticancer drugs co-delivered with the variant CD44-targeting nanoparticles can overcome the drug resistance of human CRCs. It is expected that this project may result in a novel approach to targeting TP53 and could have a major impact on the treatment of colorectal and other cancers harboring TP53 deficiency.